Project Summary The TEC family kinases are expressed in cells of hematopoietic origin and play key roles in immune cell signaling cascades during development and immune activation. Two TEC kinases, ITK and BTK are the focus of this application and are both recognized as important targets in efforts to treat immune related diseases. For example, the active site BTK inhibitor, ibrutinib, is used to treat chronic lymphocytic leukemia but new approaches are needed as drug resistance is foiling efforts to slow progression of disease. The current renewal application takes a multifaceted approach to this problem. First we are capitalizing on the mechanistic findings from the previous award period by carrying out several orthogonal screens for small molecule discovery. The findings so far suggest we have successfully identified small molecule reagents that bind to the TEC kinases and modulate T- and B-cell signaling. The screening approaches are designed to uncover allosteric modulators of kinase activity and in this way we aim to develop the means to overcome ibrutinib resistance with combination therapies as well as generally define alternative ? outside of the active site - approaches to kinase inhibition. In additional research objectives, we use our arsenal of biophysical tools (NMR spectroscopy, hydrogen/deuterium exchange mass spectrometry, and x-ray crystallography) in addition to biochemical and cellular assays to understand how mutations that drive drug resistence or oncogenicity alter the structure, dynamics and function of the full-length ITK and BTK kinases. The molecular level knowledge that will emerge from this work will provide a better understanding of T cell and B cell signaling and the means to target specific interactions or allosteric regulatory mechanisms for therapeutic uses.